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The frequent acquisition of genomic abnormalities in human preimplantation embryos is a leading cause of pregnancy loss, but does not necessarily prohibit healthy offspring. However, the impact of genomic abnormalities on cellular states and development of the early human embryo remains largely unclear. Here, we characterise aneuploidy and reconstruct gene regulatory networks in human preimplantation embryos, and investigate gene expression and developmental perturbations instigated by aneuploidy using single-cell genome-and-transcriptome sequencing (G&T-seq). At the genomic level, we show that acquired numerical and structural chromosomal aberrations are frequent across all stages of early embryogenesis and in all cell lineages. At the transcriptome level, we identify regulators of cell identity and uncover a network of 248 transcription factors from 10 major gene regulatory modules that characterise the distinct lineages of human preimplantation embryos. By integrating single-cell DNA-with RNA-information, we unveil how expression levels are affected by losses or gains of the corresponding genes in embryonic cells across human preimplantation development, as well as how copy-number aberrant transcription factor genes perturb the expression of their cognate target genes in euploid regions. Furthermore, we reveal a majority of aneuploid cells show a developmental delay and reduced fitness, indicating cell competition within the mosaic diploid-aneuploid embryo, which may contribute to selection against aneuploid cells and the birth of healthy offspring from mosaic diploid-aneuploid embryos. In summary, our multi-modal analyses provide unprecedented insights into early human embryo development.

Background Morphometric and morphokinetic evaluation of in vitro cultured human embryos allows evaluation without time restriction and reduces intra- and inter-observer variability. Even though these technologies have been reported to improve the quality of cleavage stage embryo evaluation during fresh culture, possible advantages in the evaluation of cryopreserved embryos have been scarcely explored. This study aims to compare morphometric and morphokinetic parameters between slow frozen and vitrified embryos and to determine their relationship to embryo survival and implantation rate (IR) after thawing/warming. Methods During fresh culture, morphometric characteristics (Total Cell Volume (TCV), symmetry, fragmentation and number of blastomeres) were measured in 286 thawed/warmed embryos. Likewise, after thawing/warming, similar morphometric characteristics were measured in 135 survived embryos. Moreover, morphokinetic parameters (time to mitosis resumption and time to compaction) were measured in 90 embryos after thawing/warming. Then, using linear regression, we investigated the differences between vitrified and slow frozen embryos and the relation of the measured characteristics to embryo survival and IR. Statistical corrections were applied to account for data clustering and for multiple testing. Results Vitrified embryos resume mitosis and start compaction significantly earlier than slow frozen embryos. Mitosis resumption rate was 82% for vitrified and 63% for slow frozen embryos and median time to mitosis resumption was 7.6 h and 13.1 h (p = 0.02), respectively. Compaction rate was 62% in vitrified and only 23% in slow frozen embryos. Median time to compaction was 18.1 h for vitrified embryos but, for slow frozen could not be computed since less than half of the slow frozen embryos reached compaction (p = 0.0001). Moreover, intact embryos resume mitosis significantly earlier than not intact ones regardless of the freezing method (rate: 79% vs. 66%, median time: 7.6 h vs 14.6 h, respectively, p = 0.03). Regarding morphometrics, slow frozen embryos showed lower TCV and higher blastomere symmetry after thawing than vitrified embryos despite having similar blastomere number. IR was related to blastomere number at cryopreservation in slow frozen embryos, but not in vitrified ones. Conclusions Interestingly, vitrified/warmed embryos undergo mitosis resumption and compaction significantly earlier than slow frozen/thawed embryos. However, the clinical use of this morphokinetic parameters still remains to be investigated in larger studies. Trial registration Retrospectively registered on December 15, 2015 NCT02639715 .

Background Characteristics routinely used to evaluate embryo quality after thawing include number of blastomeres survived and presence of mitosis resumption after overnight culture. It is unknown to which extent symmetry and fragmentation affect implantation after warming and whether application of stricter criteria either before vitrification or after warming would improve implantation rate (IR) of vitrified/warmed embryos. This study aimed to find new parameters to improve selection criteria for vitrification and for transfer after warming. Methods Firstly, we evaluated standard morphological characteristics (intact survival, mitosis resumption, number of blastomeres, symmetry and fragmentation) of 986 warmed day 3 embryos and, from a subset of 654, we evaluated morphometric characteristics (fragmentation, symmetry and volume change). Secondly, we tested the hypothesis that IR of day 3 vitrified/warmed embryos is influenced by morphometric characteristics. IR per embryo transferred was calculated using embryos that were transferred in a single embryo transfer (SET) or a double embryo transfer (DET) with either 0 or 100 % implantation (830/986). We investigated the significant differences in IR between the different categories of a specific characteristic. These categories were based on our standard embryo evaluation system. The statistical tests Chi-square, Fisher’s exact or Cochrane-Armitage were used according to the type and/or categories of the variable. Results The 986 embryos were transferred in 671 FET cycles with 16.9 % (167/986) IR. After exclusion of DET with 1 embryo implanted, IR per embryo transferred was 12.4 % (103/830). Embryo symmetry, fragmentation and volume change in vitrified/warmed day 3 embryos were not associated with IR. However, when mitosis resumption was present after overnight culture, intact embryos reached significantly higher IR than non-intact embryos and only when the embryo compacted after overnight culture the number of cells damaged after warming had no effect on IR. Concretely, embryos with 8 cells after warming or >9 cells after overnight culture–including compacted embryos–reached the highest IR (>15 %) while embryos with 10 % and/or symmetry

Most human pre-implantation embryos are mosaics of euploid and aneuploid cells. To determine the fate of aneuploid cells and the developmental potential of mosaic embryos, here we generate a mouse model of chromosome mosaicism. By treating embryos with a spindle assembly checkpoint inhibitor during the four- to eight-cell division, we efficiently generate aneuploid cells, resulting in embryo death during peri-implantation development. Live-embryo imaging and single-cell tracking in chimeric embryos, containing aneuploid and euploid cells, reveal that the fate of aneuploid cells depends on lineage: aneuploid cells in the fetal lineage are eliminated by apoptosis, whereas those in the placental lineage show severe proliferative defects. Overall, the proportion of aneuploid cells is progressively depleted from the blastocyst stage onwards. Finally, we show that mosaic embryos have full developmental potential, provided they contain sufficient euploid cells, a finding of significance for the assessment of embryo vitality in the clinic.

Background. Single-cell micro-metastases of solid tumors often occur in the bone marrow. These disseminated tumor cells (DTCs) may resist therapy and lay dormant or progress to cause overt bone and visceral metastases. The molecular nature of DTCs remains elusive, as well as when and from where in the tumor they originate. Here, we apply single-cell sequencing to identify and trace the origin of DTCs in breast cancer. Results. We sequence the genomes of 63 single cells isolated from six non-metastatic breast cancer patients. By comparing the cells’ DNA copy number aberration (CNA) landscapes with those of the primary tumors and lymph node metastasis, we establish that 53% of the single cells morphologically classified as tumor cells are DTCs disseminating from the observed tumor. The remaining cells represent either non-aberrant ‘normal’ cells or ‘aberrant cells of unknown origin’ that have CNA landscapes discordant from the tumor. Further analyses suggest that the prevalence of aberrant cells of unknown origin is age-dependent, and that at least a subset is hematopoietic in origin. Evolutionary reconstruction analysis of bulk tumor and DTC genomes enables ordering of CNA events in molecular pseudo-time and traced the origin of the DTCs to either the main tumor clone, primary tumor subclones, or subclones in an axillary lymph node metastasis. Conclusions. Single-cell sequencing of bone marrow epithelial-like cells, in parallel with intra-tumor genetic heterogeneity profiling from bulk DNA, is a powerful approach to identify and study DTCs, yielding insight into metastatic processes. A heterogeneous population of CNA-positive cells is present in the bone marrow of non-metastatic breast cancer patients, only part of which are derived from the observed tumor lineages. Citation Format: Jonas Demeulemeester, Parveen Kumar, Elen K. Møller, Silje Nord, David C. Wedge, April Peterson, Randi R. Mathiesen, Renathe Fjelldal, Masoud Z. Esteki, Koen Theunis, Elia F. Gallardo, Jason Grundstad, Elin Borgen, Lars O. Baumbusch, Anne-Lise Børresen-Dale, Kevin P. White, Vessela N. Kristensen, Peter Van Loo, Thierry Voet, Bjørn Naume. Tracing the origin of disseminated tumor cells in breast cancer using single-cell sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3049. doi:10.1158/1538-7445.AM2017-3049